Hyaluronic acid was discovered by Meyer and Palmer in 1934. Karl Meyer isolated the polysaccharide from the vitreous humor. Since it contained uronic acid, Meyer named the substance hyaluronic acid from hyalos (meaning glassy, vitreous) and uronic acid. At physiological pH all carboxyl groups on the uronic acid residue are dissociated and the polysaccharide was named sodium hyaluronate when sodium is the counter ion. In 1986, Balazs suggested the name hyaluranon. This is currently the accepted terminology. The abbreviation “HA” will be used in this application to designate hyaluranon, which includes hyaluronic acid and its metallic salts.
HA is a linear polysaccharide (long-chain biological polymer) formed by repeating disaccharide units consisting of D-glucuronic acid and N-acetyl-D-glucosamine linked by β(1-3) and β(1-4) glycosidic linkages. HA is distinguished from the other glycosaminoglycans, as it is free from covalent links to protein and sulphuric groups. It is however an integral component of complex proteoglycans. HA is an important component of the intercellular matrix, the material filling the space between the cells of such diverse tissues as skin, tendons, muscles and cartilage.
HA has been discovered as a coat attached to cell surface, as part of large molecular structures, and as free polysaccharide, e.g., in synovial fluid and in the vitreous body. HA is ubiquitous in animals, with the highest concentration found in soft connective tissue. It plays an important role for both mechanical and transport purposes in the body; e.g., it gives elasticity to the joints and rigidity to the vertebrate disks, and it is also a constituent of major importance in the vitreous body of the eye.
HA, because of its high degree of hydration, is probably responsible for the high content of water of some tissues, increasing the resistance of such tissues to compression. This is a role based on hyaluronic acid's capacity to hold more water than any other natural or synthetic polymer. The size (MW) of individual molecules and the potential for intermolecular interactions determines whether a particular HA preparation will form an elastoviscous matrix under given conditions.
The large molecular volume forces the overlap of individual HA molecular domains, resulting in extensive chain entanglement and chain-chain interaction. It is the resulting intertwined polymeric network, which acts as the jelly-like milieu supporting and influencing tissue functions.
Hyaluronic acid serves as a thickening agent to synovial fluid, and the viscoelastic effect is such that under impact a hyaluronic acid solution is hard elastic, but under slow movement the viscosity effect is more operable. The shock absorber effect is particularly important and effective in young people.
HA is a natural lubricant and synovial fluid requires the presence of hyaluronic acid to be an effective lubricant of the synovial membrane. The network-making property plays a primary role in the connective tissue; this role differs from those of the chondroitin sulphate, dermatan sulphate and keratan sulphate, their water retention abilities and, therefore, their solution viscosity being inferior.
Hyaluronic acid may also play an important role in the control of intercellular and interstitial permeability. This theory is supported by the fact that the depolymerization of hyaluronic acid, which occurs in certain pathological conditions of connective tissues, results in an increased permeability of the connective tissue barrier.
Hyaluronic acid is involved in ossification processes. Calcium (II) ions can become strongly associated with a hyaluronic acid proteoglycan and are not readily displaced, even by high concentrations of univalent ions. There is evidence for a role of proteoglycans in regulation of mineral phase separation in calcifying cartilage and calcification itself.
The intraarticular injection of viscoelastic high molecular weight (HMW)-HA solutions provides protection, lubrication, pain reduction and hydration to the articular cartilage and capsular tissues. This injection may restore the damaged HA layer on the articular cartilage surface, bringing about an alleviation of arthritic condition and arrest of the process of disease.
The HA properties are dependent on the molecular weight, the solution concentration, and physiological pH. In low concentrations, the individual chains entangle and form a continuous network in solution, which gives the system interesting properties, such as pronounced viscoelasticity and pseudoplasticity that is unique for a water-soluble polymer at low concentration.
HA exhibits viscous flow, elastic and pseudoplastic properties. This property is unique to HA. Other glycosaminoglycans, GAGs, may form viscous solutions, but only at considerably greater concentrations than HA, and they never form a viscoelastic polymer network. HA has been demonstrated to be important in different activities such as tissue hydration, lubrication, solute transportation, cell migration, cell function, cell differentiation, and cell proliferation.
Sources and Manufacturing of HA: During the 1930s and 1940s Meyer and others isolated hyaluranon from a number of sources and relatively large amounts were found in vitreous, synovial fluid, umbilical cord, skin, and rooster comb. HA has historically been isolated from synovial fluid, umbilical cord, skin, and rooster comb. However, Kendall in 1937 isolated HA from certain strains of bacteria, such as streptococci. Today HA is also obtained from bacterial fermentation.
The isolation of HA from rooster combs typically includes the following steps: An enzymatic digestion, a specific separation in order to remove protein and a purification to provide a crude extract. Further purification steps include precipitation in ethanol and redissolution in sodium chloride solution. Thus, a typical process for isolating HA from rooster comb includes removal of epithelium from the combs, grinding of combs, treatments in acetone and multiple treatments with ethanol and sodium chloride solutions. Several U.S. patents describe methods to isolate and purify hyaluronic acid including U.S. Pat. Nos. 4,141,973; 4,784,990; 5,099,013; 5,166,331; 5,316,926; 5,411,874; 5,559,104 and 5,925,626, which are incorporated herein by reference.
There are some differences between the isolation-origin and the fermentation-origin HA. The HA obtained from isolation methods has the natural structure of the glycosaminoglycan (formally called mucopolysaccharide) in natural tissues. In this sense it is much more porous than that obtained by fermentation, and therefore can exhibit great differences of dispersability in water. HA obtained by bacterial fermentation typically includes higher levels of bacteria and, when cultured, has a higher number of colony forming units (c.f.u.) than HA obtained by isolation methods, due to the high level of nutrients in the fermentation media. Further, fermentation-origin HA typically includes significant levels of endotoxins that must be removed. The HA obtained by fermentation also needs to be more purified in order to eliminate as many bacterial proteins as possible.
There are also drawbacks with HA prepared by many of the known methods or from known sources. These drawbacks can include, for example, an inflammatory response when used in compositions for treating mammals and postoperative complications in ocular surgery. Although, many of the drawbacks can be avoided or minimized by additional processing, it results in an increased costs in obtaining HA having the requisite purity and functionality.
Although HA obtained from known sources, discussed above, have found use in connection with humans and animals, e.g., as ingredients for cosmetics, pharmaceuticals and nutraceuticals, these known sources contain relatively low levels of HA or require significant processing to produce HA in the required purity. This also results in a relatively high cost for useable HA.
The composition of egg and/or eggshell membranes, as well as the eggshell, have also been previously reported. Eggshell membranes are composed of protein fibers between the albumin and the inner surface of the shell. The proteins have a high concentration of arginine, glutamic acid, methionine, histidine, cystine, and proline.1 Additional investigations of eggshell membrane have demonstrated the presence of high concentrations of collagen, including Type I and Type X collagen.2,3 Further investigations have identified keratan and dermatan sulfate in eggshell.4 Studies have also reported the absence of uronic acid in eggshell membrane.5 One study, which investigated the acid glycosaminoglycan content of the isthmus region of hen oviduct and the egg membrane of shell-free eggs, reported very low levels of hyaluronic acid in the egg membrane collected from the shell-free eggs.6 However, neither the presence of significant quantities of hyaluronic acid (HA) in eggshell membrane nor the extraction of HA from eggshell membrane have been previously reported.
Thus, there is a need for new sources of HA, which have higher concentrations of the naturally occurring HA. There is also a need for HA derived from new sources, which does not have the problems associated with known sources of HA, requires less processing and/or results in more cost effective methods for obtaining useful HA products.